Multivalent feline vaccine

ABSTRACT

The present invention provides new multivalent vaccines for felines. The present invention also provides methods of making and using the multivalent vaccines alone or in combinations with other protective agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No. 16/760,160, filed on Apr. 29, 2020, which is a national stage entry under 35 U.S.C. § 371 of PCT/EP2018/080106, filed on Nov. 5, 2018, which claims priority under 35 U.S.C. § 119(e) of provisional application U.S. Ser. No. 62/599,401 filed Dec. 15, 2017, 62/596,508 filed Dec. 8, 2017, U.S. Ser. No. 62/582,050, filed Nov. 6, 2017, and U.S. Ser. No. 62/581,955 filed Nov. 6, 2017, the contents of which are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to new multivalent vaccines for felines. Methods of making and using the multivalent vaccines alone or in combination with other protective agents are also provided.

BACKGROUND

Feline respiratory disease includes those illnesses typified by rhinosinusitis, conjunctivitis, lacrimation, salivation, and oral ulcerations. The two most common pathogens associated with upper respiratory disease in cats are feline calicivirus (FCV) and feline viral rhinotracheitis virus (FVR), which is also known as feline herpesvirus type 1 virus (FHV-1). These two feline viruses are considered responsible for approximately 80% of all feline respiratory disease, worldwide. A bacterium, Chlamydophila felis, is a third pathogen that also can play a role in feline respiratory disease. Accordingly, vaccines against these three pathogens are now commercially available.

FVR is an alphaherpesvirus that is related to canine herpesvirus-1 and is perhaps the most important of the cat respiratory pathogens. FVR is a large, enveloped DNA virus that is extremely contagious and can lead to severe disease in kittens, as well as cats. Accordingly, most cats are exposed to FVR during their lifetime. The commercially available Nobivac® Feline-1 vaccine contains a modified live feline viral rhinotracheitis virus.

The most common characteristic and clinical signs of FCV infection are the development of vesicles (ulcers) on the tongue and oral mucosa, which begin as small, individual, ulcers but can spread and affect a large part of the tongue. Fever often also is observed in infected cats. Certain strains of FCV also cause a disease in cats known as limping syndrome, which is characterized by fever, joint and muscle soreness (limping), and occasional lingual/oral ulceration. In addition, some strains of FCV have been associated with chronic stomatitis in infected cats. Cats infected with FCV may become persistently infected, and may shed infectious virus for long periods of time.

FCV isolates are antigenically highly variable, and antibodies from cats vaccinated with older vaccine strains of FCV, such as FCV F9, do not efficiently neutralize all current field isolates. Moreover, new FCV strains associated with systemic disease and high mortality have been identified [see e.g., U.S. Pat. No. 7,449,323 B2]. These “virulent systemic” (VS-FCV) isolates are responsible for localized outbreaks, and current vaccines also do not appear to protect cats from disease caused by these strains.

FCV comprises a single-stranded, positive-sense RNA genome consisting of three open reading frames (ORFs). The genome is polyadenylated at the 3′ end and bound by a virally-encoded protein at the 5′-end. The first open reading frame encodes a viral protease and an RNA-dependent RNA polymerase, which are expressed on a single polypeptide. This polypeptide then is post-translationally cleaved by the viral protease. The second open reading frame encodes the major capsid protein (i.e., the FCV capsid protein), which has six regions denoted as A-F [Scott et al., 60 Am. J. Vet. Res.:652-658 (1999)]. Region A is cleaved to produce the mature capsid protein. Whereas regions B, D, and F of ORF2 are relatively conserved between FCV isolates, regions C and E are variable, with region E of ORF2 containing the major B-cell epitopes [see, Radford et al., 38(2) Vet Res.:319-335 (2007)]. ORF 3 encodes a minor structural protein [Sosnovtsev and Green, 277 Virology: 193-203 (2000)].

Chlamydophila felis is a bacterium that is endemic among domestic cats worldwide. C. felis can lead to inflammation of conjunctiva, rhinitis, and respiratory problems in the infected cat C. felis has a relatively small genome that encodes only about a thousand proteins. This bacterium also contains a plasmid comprising 75,000 base pairs. The commercially available Nobivac® Feline-1 vaccine contains a modified live Chlamydophila felis.

In addition to the three pathogens related to feline respiratory disease discussed above, cats often also are vaccinated against three other viruses: feline leukemia virus (FeLV), feline panleukopenia (FPV or FPLV), and rabies virus. Feline leukemia virus is a retrovirus that infects domestic cats, resulting in significant morbidity and mortality worldwide. Though predominantly transmitted through saliva, FeLV has been reported also to be spread through contact with body fluids [Pacitti et al., Vet Rec 118:381-384 (1986) doi:10.1136/vr.118.14.381, Levy et al., J Feline Med Surg 10:300-316 (2008) doi:10.1016/j.jfms.2008.03.002]. The clinical signs in cats observed during FeLV infections include: cytoproliferative disorders (lymphoid or myeloid tumors), cytosuppressive disorders (infectious diseases associated with immunosuppression, anemia, myelosuppression), inflammatory disorders, neurological disorders, abortions, and enteritis [Hoover et al., J Am Vet Med Assoc 199:1287-1297 (1991); Levy and Crawford, Textbook of Veterinary Internal Medicine, 6th ed (Ettinger S J, Feldman E C., eds.) WB Saunders, Philadelphia, Pa. (2005)]. The prevalence of antigenemia may vary from 1-5% in healthy cats to 15-30% in afflicted cats [Hosie et al. Veterinary Records, 128: 293-297 (1989); Braley, Feline Practice 22: 25-29 (1994); Malik et al., Australian Veterinary Journal 75:323-327 (1997); Arjona et al., Journal of Clinical Microbiology 38:3448-3449 (2000)]. FeLV frequently establishes a lasting infection with a concomitant persistent viremia, often leading to the death of the host cat.

The single stranded RNA genome of FeLV encodes only three genes: (i) an ENV gene, which encodes the envelope glycoprotein, (ii) a GAG gene, which encodes the major structural components of the virus, and (iii) a POL gene, which encodes the RNA polymerase [Thomsen et al., Journal of General Virology 73:1819-1824 (1992)]. The FeLV envelope (ENV) gene encodes a gp85 precursor protein which is proteolytically processed by one or more cellular enzymes to yield the major envelope glycoprotein gp70 and the associated transmembrane protein p15E [DeNoronha, et al., Virology 85:617-621 (1978); Nunberg et al., PNAS 81:3675-3679 (1983)]. The transmembrane protein p15E contains a sequence conserved among gammaretroviruses with immunosuppressive properties [Mathes et al., Nature 274:687-689 (1978)]. Recently, The European Medicines Agency's Committee for Medicinal Products for Veterinary Use (CVMP) has adopted a positive opinion for a vaccine comprising a recombinant p45 FeLV-envelope antigen derived from the gp70 surface glycoprotein of the FeLV subgroup A that is expressed in Escherichia coli as active substance. The FeLV envelope glycoprotein is the target of FeLV-specific cytotoxic T cell responses, as well as neutralizing antibodies and accordingly, one of the major immunogens of FeLV [Flynn et al., J. Virol. 76(5): 2306-2315 (2002)].

Feline panleukopenia (FPV or FPLV) is a highly contagious viral disease of cats that is often fatal. The name panleukopenia derives from the low white blood cell count (leucocytes) exhibited by affected animals. FPLV infects and destroys actively dividing cells in lymphoid tissues, bone marrow, intestinal epithelium, and in very young kittens, the retina and cerebellum. The virus also may spread in pregnant cats transplacentally to cause embryonic resorption, fetal mummification, stillbirth, or abortion. Infected cats shed the virus in their urine, stool, and nasal secretions resulting in infection in susceptible cats when they come in contact with these secretions or fleas from infected cats. The clinical signs due to an FPLV infection have also been labeled as feline distemper or feline parvo.

Feline panleukopenia virus is a member of the genus Parvovirus, in the family Parvoviridae. Accordingly, FPLV is closely related to mink enteritis virus and canine Type 2 parvovirus (CPV-2). FPLV has a single stranded DNA genome that has been sequenced [see, Liu et al., Genome Announc. March-April; 3(2) (2015): e01556-14]. The commercially available Nobivac® Feline-1 vaccine contains a modified live feline panleukopenia virus.

Rabies is a preventable zoonotic disease that leads to inflammation of the brain in humans and other mammals. Clinical rabies is an acute, progressive encephalitis that is typically classified as either furious or paralytic rabies. Furious rabies is characterized by restlessness, irritability and aggression. Paralytic rabies is characterized by excessive salivation, deep, labored breathing, paralysis, and eventually coma. The causative agent of rabies is the rabies virus, which is capable of infecting most mammals, including felines, and maintains a reservoir of disease in wild and susceptible domestic animals.

The rabies virus is an enveloped, RNA virus that encodes five structural proteins: a nucleoprotein (N), a phosphoprotein (P), a matrix protein (M), a glycoprotein (G), and an RNA-dependent RNA polymerase [Dietzschold et al., Crit Rev Immunol 10:427-439 (1991)]. The glycoprotein (G) is considered the protective antigen which induces virus neutralizing antibodies [Cox et al., Infect Immun 16:754-759 (1977)]. Several types of rabies vaccines have been produced to combat this disease. Inactivated cell culture derived whole-virus killed rabies virus vaccines are the most commonly used vaccines in the United States. These whole-virus killed rabies virus vaccines require high levels of antigen and therefore, require an adjuvant. Unfortunately, this use of an adjuvant is associated with injection site reactivity, hypersensitivity, and even with the perceived risk of injection site sarcomas in cats. Recently, a modified live vaccine has been used successfully with oral vaccine baits for the immunization of wild animals [Mahl et al., Vet Res 45(1):77 (2014)]. In addition, a recombinant vaccine expressing the glycoprotein (G) is currently being marketed in the United States for use in cats. Nucleic acid vaccines also have been used in laboratory studies, though none are currently licensed in the United States.

A number of vector strategies have been employed through the years for vaccines in an effort to protect against certain pathogens. One such vector strategy includes the use of alphavirus-derived replicon RNA particles (RP) [Vander Veen, et al. Anim Health Res Rev. 13(1):1-9. (2012) doi: 10.1017/S1466252312000011; Kamrud et al., J Gen Virol. 91(Pt 7):1723-1727 (2010)] which have been developed from several different alphaviruses, including Venezuelan equine encephalitis virus (VEE) [Pushko et al., Virology 239:389-401 (1997)], Sindbis (SIN) [Bredenbeek et al., Journal of Virology 67:6439-6446 (1993)], and Semliki Forest virus (SFV) [Liljestrom and Garoff, Biotechnology (NY) 9:1356-1361 (1991)]. RP vaccines deliver propagation-defective alphavirus RNA replicons into host cells and result in the expression of the desired antigenic transgene(s) in vivo [Pushko et al., Virology 239(2):389-401 (1997)]. RPs have an attractive safety and efficacy profile when compared to some traditional vaccine formulations [Vander Veen, et al. Anim Health Res Rev. 13(1):1-9. (2012)]. The RP platform has been used to encode pathogenic antigens and is the basis for several USDA-licensed vaccines for swine and poultry.

Notably, alphavirus RNA replicon particles, and in particular Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particles, have been reported to catalyze a systemic antiviral state and protect against lethal virus challenge [Konopka et al., J. Virol., 83 (29):12432-12442 (2009)] and more particularly, induce rapid protection against foot-and-mouth disease virus [Segundo et al., J. Virol., 87 (10):5447-5460 (2013)]. Accordingly, because alphavirus RNA replicon particles enhance the innate immune response against live virus it would appear that they should not be combined with a modified live virus in a vaccine because it would be expected to be detrimental to the immune response.

The citation of any reference herein should not be construed as an admission that such reference is available as “prior art” to the instant application.

SUMMARY OF THE INVENTION

Accordingly, the present invention includes immunogenic compositions that comprise alphavirus RNA replicon particles that encode one or more antigens from one or more feline pathogens along with one or more modified live feline pathogens. All of the immunogenic compositions of the present invention also may be used in multivalent vaccines. In a particular embodiment of this type, the vaccinated subject is a feline. In a more particular embodiment, the vaccinated subject is a domestic cat. Methods of making and using the immunogenic compositions and vaccines of the present invention are also provided.

In particular embodiments, the immunogenic compositions comprise alphavirus RNA replicon particles that encode one or more feline calicivirus (FCV) antigens and a modified live feline pathogen. In other embodiments, the immunogenic compositions comprise alphavirus RNA replicon particles that encode one or more feline leukemia virus (FeLV) antigens and a modified live feline pathogen. In yet other embodiments, the immunogenic compositions comprise alphavirus RNA replicon particles that encode one or more rabies virus antigens and a modified live feline pathogen. In certain embodiments, the modified live feline pathogen is a feline viral rhinotracheitis virus (FVR). In other embodiments, the modified live feline pathogen is a feline panleukopenia virus (FPLV). In yet other embodiments, the modified live feline pathogen is a modified live Chlamydophila felis. In still other embodiments, the modified live feline pathogen is a modified live F9-like feline calicivirus (FCV F9-Like). In yet other embodiments, the modified live feline pathogen is a modified live Bordetella bronchiseptica. The present invention further provides immunogenic compositions that comprise any combination of these alphavirus RNA replicon particles and modified live feline pathogens. In specific embodiments of this type, the immunogenic composition comprises alphavirus RNA replicon particles encoding an FCV antigen, alphavirus RNA replicon particles encoding an FeLV antigen, a modified live FVR, a modified live FPLV, and a modified live Chlamydophila felis.

In certain embodiments of this type, the immunogenic compositions comprise alphavirus RNA replicon particles that encode an FCV capsid protein. In more particular embodiments, the FCV capsid protein is an FCV F9-Like capsid protein. In other embodiments, the alphavirus RNA replicon particle encodes an antigenic fragment of an FCV F9-Like capsid protein. In yet other embodiments, the FCV capsid protein is a virulent systemic FCV (VS-FCV) capsid protein. In still other embodiments, the alphavirus RNA replicon particle encodes an antigenic fragment of a VS-FCV capsid protein. In yet other embodiments, the alphavirus RNA replicon particle encodes both an FCV F9-Like capsid protein or antigenic fragment thereof, and an VS-FCV capsid protein or antigenic fragment thereof.

In other embodiments, the immunogenic compositions comprise alphavirus RNA replicon particles that encode a VS-FCV capsid protein or antigenic fragment thereof in which the VS-FCV capsid protein comprises an amino acid sequence comprising 95% identity or more with the amino acid sequence of SEQ ID NO: 2. In more specific embodiments, the VS-FCV capsid protein comprises the amino acid sequence of SEQ ID NO: 2. In even more specific embodiments, the VS-FCV capsid protein is encoded by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 12. In related embodiments, an alphavirus RNA replicon particle of the present invention encodes a FCV F9-Like capsid protein or antigenic fragment thereof. In specific embodiments of this type, the FCV F9-Like capsid protein comprises an amino acid sequence comprising 95% identity or more with the amino acid sequence of SEQ ID NO: 4. In more specific embodiments, the FCV F9-Like capsid protein comprises the amino acid sequence of SEQ ID NO: 4. In even more specific embodiments of this type, the FCV F9-Like capsid protein is encoded by the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 13.

In related embodiments, the immunogenic compositions comprise alphavirus RNA replicon particles that encode one or more feline leukemia virus (FeLV) antigens. In certain embodiments, the FeLV antigen is a FeLV glycoprotein (e.g., gp85). In other embodiments, the alphavirus RNA replicon particle encodes an antigenic fragment of the FeLV gp85. In more specific embodiments of this type, the antigenic fragment of the FeLV glycoprotein is FeLV gp70. In a related embodiment, the antigenic fragment of the FeLV glycoprotein is FeLV gp45. In more specific embodiments of this type, the FeLV gp85 comprises an amino acid sequence comprising 95% identity or more with the amino acid sequence of SEQ ID NO: 6. In more specific embodiments of this type, the FeLV gp85 comprises the amino acid sequence of SEQ ID NO: 6. In even more specific embodiments of this type, the FeLV gp85 is encoded by the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 14. In related embodiments, the FeLV gp70 comprises an amino acid sequence comprising 95% identity or more with the amino acid sequence of SEQ ID NO: 8. In more specific embodiments of this type, the FeLV gp70 comprises the amino acid sequence of SEQ ID NO: 8. In even more specific embodiments of this type, the FeLV gp70 is encoded by the nucleotide sequence of SEQ ID NO: 7 or SEQ ID NO: 15.

In yet other embodiments the immunogenic compositions comprise alphavirus RNA replicon particles that encode a rabies virus glycoprotein (G). In still other embodiments, the alphavirus RNA replicon particles encode an antigenic fragment of the rabies virus G. In more specific embodiments of this type, the rabies virus G comprises an amino acid sequence comprising 95% identity or more with the amino acid sequence of SEQ ID NO: 10. In still more specific embodiments of this type, the rabies virus G comprises the amino acid sequence of SEQ ID NO: 10. In even more specific embodiments of this type, the rabies virus G is encoded by the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 16.

In particular embodiments, the alphavirus RNA replicon particle is a Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particle. In more specific embodiments of this type, the VEE alphavirus RNA replicon particle is a TC-83 VEE alphavirus RNA replicon particle. In other embodiments, the alphavirus RNA replicon particle is a Sindbis (SIN) alphavirus RNA replicon particle. In still other embodiments, the alphavirus RNA replicon particle is a Semliki Forest virus (SFV) alphavirus RNA replicon particle. In an alternative embodiment, a naked DNA vector encodes one or more antigens that originate from one or more feline pathogens. In particular embodiments of this type, the naked DNA vectors encode an FCV capsid protein or antigenic fragment thereof. In specific embodiments of this type, the naked DNA vectors encode an VS-FCV capsid protein or antigenic fragment thereof. In other embodiments of this type, the naked DNA vectors encode an FeLV gp85, or antigenic fragment thereof.

In certain embodiments, the immunogenic compositions can include at least one modified live feline pathogen and alphavirus RNA replicon particles that encode one or more FCV capsid proteins or antigenic fragments thereof, one or more FeLV glycoproteins or antigenic fragments thereof, and/or one or more rabies virus G proteins or antigenic fragments thereof. In particular embodiments of this type, the alphavirus RNA replicon particles encode both a VS-FCV capsid protein or an antigenic fragment thereof, and an FCV F9-Like capsid protein or an antigenic fragment thereof. In related embodiments, the alphavirus RNA replicon particles encode both a VS-FCV capsid protein or an antigenic fragment thereof, and an FeLV gp85 or an antigenic fragment thereof. In other embodiments, the alphavirus RNA replicon particles encode both a FCV F9-Like capsid protein or an antigenic fragment thereof, and an FeLV gp85 or an antigenic fragment thereof. In alternative embodiments, the alphavirus RNA replicon particles encode both a VS-FCV capsid protein or an antigenic fragment thereof, and a rabies virus G protein or an antigenic fragment thereof. In yet other embodiments, the alphavirus RNA replicon particles encode both a FCV F9-Like capsid protein or an antigenic fragment thereof, and a rabies virus G protein or an antigenic fragment thereof. In still other embodiments, the alphavirus RNA replicon particles encode a VS-FCV capsid protein or an antigenic fragment thereof, an FeLV gp85 or an antigenic fragment thereof, and a rabies virus G protein or an antigenic fragment thereof. In alternative embodiments, the alphavirus RNA replicon particles encode a VS-FCV capsid protein or an antigenic fragment thereof, a FCV F9-Like capsid protein or an antigenic fragment thereof, and an FeLV gp85 or an antigenic fragment thereof. In yet other embodiments, the alphavirus RNA replicon particles encode a VS-FCV capsid protein or an antigenic fragment thereof, an FeLV gp85 or an antigenic fragment thereof, a rabies virus G protein or an antigenic fragment thereof, and an FCV F9-Like capsid protein or an antigenic fragment thereof. In still other embodiments, the alphavirus RNA replicon particles encode an FeLV gp85 or an antigenic fragment thereof, and a rabies virus G protein or an antigenic fragment thereof. In yet other embodiments, the alphavirus RNA replicon particles encode an FeLV gp85 or an antigenic fragment thereof, an FCV F9-Like capsid protein or an antigenic fragment thereof, and a rabies virus G protein or an antigenic fragment thereof.

Accordingly, the present invention provides immunogenic compositions that include at least one modified live feline pathogen and any one or more of the alphavirus RNA replicon particles of the present invention that encode multiple feline pathogen antigens, and/or any one or more alphavirus RNA replicon particles of the present invention that encode a single feline pathogen antigen. In particular embodiments, the modified live feline pathogen is a modified live FVR. In other embodiments, the modified live feline pathogen is a modified live FPLV. In yet other embodiments, the modified live feline pathogen is a modified live Chlamydophila felis. In still other embodiments, the modified live feline pathogen is a modified live FCV F9-Like. In yet other embodiments, the modified live feline pathogen is a modified live Bordetella bronchiseptica. In particular embodiments, two or more modified live feline pathogens are included in the immunogenic composition. In specific embodiments of this type, the immunogenic composition comprises a modified live FVR and a modified live Chlamydophila. In other embodiments, three or more modified live feline pathogens are included in the immunogenic composition. In specific embodiments of this type, the immunogenic composition comprises a modified live FVR, a modified live Chlamydophila, and a modified live feline pathogen is an FPLV. In still other embodiments, four or more modified live feline pathogens are included in the immunogenic composition. In specific embodiments of this type, the immunogenic composition comprises a modified live FVR, a modified live Chlamydophila, a modified live FPLV, and a modified live F9-Like FCV. In particular embodiments, all of the alphavirus RNA replicon particles in the immunogenic composition are Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particles. In yet more specific embodiments, all of VEE alphavirus RNA replicon particles in the immunogenic compositions are TC-83 VEE alphavirus RNA replicon particles.

In additional embodiments, alphavirus RNA replicon particles can encode protein antigens (or antigenic fragments thereof) that originate from other feline pathogens. In particular embodiments of this type, the protein antigen originates from a feline pneumovirus (FPN). In still other embodiments, the protein antigen originates from feline parvovirus (FPV). In yet other embodiments, the protein antigen originates from feline infectious peritonitis virus (FIPV). In yet other embodiments, the protein antigen originates from feline immunodeficiency virus. In still other embodiments, the protein antigen originates from borna disease virus (BDV). In yet other embodiments, the protein antigen originates from feline influenza virus. In still other embodiments, the protein antigen originates from feline coronavirus (FCoV).

The present invention further provides immunogenic compositions and/or vaccines (multivalent vaccines) that include the alphavirus RNA replicon particles of the present invention together with one or more modified live (e.g., attenuated) feline pathogens of the present invention, together with a killed feline pathogen. In particular embodiments, the immunogenic compositions can further comprise a killed Chlamydophila felis, and/or killed FVR, and/or killed F9-like FCV, and/or killed VS-FCV, and/or killed FeLV, and/or killed FPLV. In certain embodiments, vaccines comprise an immunologically effective amount of one or more of these immunogenic compositions.

The present invention further comprises vaccines and multivalent vaccines that comprise the immunogenic compositions of the present invention. In particular embodiments, the multivalent vaccines are nonadjuvanted vaccines. In certain embodiments, the vaccine aids in the prevention of disease due to FCV, and/or FeLV, and/or FVR, and/or FPLV, and/or rabies virus, and/or Chlamydophila felis. In related embodiments, antibodies are induced in a feline subject when the feline is immunized with the vaccine. The present invention further includes all of the alphavirus RNA replicon particles and the naked DNA vectors of the present invention.

The present invention also provides methods of immunizing a feline against a feline pathogen, e.g., FCV, comprising administering to the feline an immunologically effective amount of a vaccine or multivalent vaccine of the present invention. In particular embodiments the vaccine is administered via intramuscular injection. In alternative embodiments the vaccine is administered via subcutaneous injection. In other embodiments the vaccine is administered via intravenous injection. In still other embodiments the vaccine is administered via intradermal injection. In yet other embodiments the vaccine is administered via transdermal injection. In still other embodiments the vaccine is administered via oral administration. In yet other embodiments the vaccine is administered via intranasal administration. In specific embodiments, the feline is a domestic cat.

The vaccines and multivalent vaccines of the present invention can be administered as a primer vaccine and/or as a booster vaccine. In specific embodiments, a vaccine of the present invention is administered as a one shot vaccine (one dose), without requiring subsequent administrations. In certain embodiments, in the case of the administration of both a primer vaccine and a booster vaccine, the primer vaccine and the booster vaccine can be administered by the identical route. In certain embodiments of this type, the primer vaccine and the booster vaccine are both administered by subcutaneous injection. In alternative embodiments, in the case of the administration of both a primer vaccine and a booster vaccine, the administration of the primer vaccine can be performed by one route and the booster vaccine by another route. In certain embodiments of this type, the primer vaccine can be administered by subcutaneous injection and the booster vaccine can be administered orally.

The invention further provides for a method of immunizing a feline against FCV comprising injecting the feline with an immunologically effective amount of the above described inventive vaccines. In particular embodiments, the vaccines can include from about 1×10⁴ to about 1×10¹⁰ RPs or higher, for example. In more particular embodiments, the vaccines can include from about 1×10⁵ to about 1×10⁹ RPs. In even more particular embodiments the vaccines can include from about 1×10⁶ to about 1×10⁸ RPs. In particular embodiments, the feline is a domestic cat.

In particular embodiments the vaccines of the present invention are administered in 0.05 mL to 3 mL doses. In more particular embodiments the dose administered is 0.1 mL to 2 mLs. In still more particular embodiments the dose administered is 0.2 mL to 1.5 mLs. In even more particular embodiments the dose administered is 0.3 to 1.0 mLs. In still more particular embodiments the dose administered is 0.4 mL to 0.8 mLs.

These and other aspects of the present invention will be better appreciated by reference to the following Detailed Description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides safe and efficacious multivalent vaccines. In particular embodiments the vaccine is nonadjuvanted. In this aspect of the invention, the vaccines do not induce feline injection-site sarcomas, yet still aid in the protection of the vaccinates from disease caused by infections by feline calicivirus (FCV) and/or feline leukemia virus (FeLV), and infections by feline viral rhinotracheitis virus (FVR), and/or feline panleukopenia virus (FPLV), and/or live Chlamydophila felis.

Despite the known enhancement of the innate immune system by alphavirus RNA replicon particles, the multivalent vaccines of the present invention, which comprise both alphavirus RNA replicon particles and modified live viruses, are unexpectedly safe and efficacious, without the alphavirus RNA replicon particles significantly interfering with the immunological effect of the modified live viruses. Indeed, alphavirus RNA replicon particles, and in particular Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particles, have previously been shown to catalyze a systemic antiviral state and protect against lethal virus challenge [Konopka et al., J. Virol., 83 (29):12432-12442 (2009)]. Moreover, VEE alphavirus RNA replicon particles have been reported to induce rapid protection against foot-and-mouth disease virus [Segundo et al., J. Virol., 87 (10):5447-5460 (2013)]. Therefore, it would have been anticipated that vaccines comprising both alphavirus RNA replicon particles and modified live viruses would lead to a substantial inhibition of the immunological effect of the modified live viruses. However, the enhancement of the innate immune response due to the presence of the alphavirus RNA replicon particles surprisingly proved not to be detrimental to the immune response induced by the accompanying modified live viruses.

Accordingly, in a particular aspect, the present invention provides vaccines that comprise alphavirus RNA replicon particles that encode an FCV capsid protein or antigenic fragment thereof and/or an FeLV gp85 or antigentic fragment thereof, along with a modified live feline FVR, and/or a modified live FPLV, and/or a modified live Chlamydophila felis. In yet another aspect of the present invention, the vaccines comprise naked DNA vectors that encode an FCV capsid protein or antigenic fragment thereof and/or an FeLV gp85 or antigentic fragment thereof, along with a modified live feline FVR, and/or a modified live FPLV, and/or a modified live Chlamydophila felis.

The vaccines of the present invention can be administered to a feline in the absence of an adjuvant and still effectively aid in the protection of the vaccinated feline against FCV.

In order to more fully appreciate the invention, the following definitions are provided.

The use of singular terms for convenience in description is in no way intended to be so limiting. Thus, for example, reference to a composition comprising “a polypeptide” includes reference to one or more of such polypeptides. In addition, reference to an “alphavirus RNA replicon particle” includes reference to a plurality of such alphavirus RNA replicon particles, unless otherwise indicated.

As used herein the term “approximately” is used interchangeably with the term “about” and signifies that a value is within fifty percent of the indicated value i.e., a composition containing “approximately” 1×10⁸ alphavirus RNA replicon particles per milliliter contains from 0.5×10⁸ to 1.5×10⁸ alphavirus RNA replicon particles per milliliter.

As used herein, the term “feline” refers to any member of the Felidae family. Domestic cats, pure-bred and/or mongrel companion cats, and wild or feral cats are all felines.

As used herein, the term “replicon” refers to a modified RNA viral genome that lacks one or more elements (e.g., coding sequences for structural proteins) that if they were present, would enable the successful propagation of the parental virus in cell cultures or animal hosts. In suitable cellular contexts, the replicon will amplify itself and may produce one or more sub-genomic RNA species.

As used herein, the term “alphavirus RNA replicon particle”, abbreviated “RP”, is an alphavirus-derived RNA replicon packaged in structural proteins, e.g., the capsid and glycoproteins, which also are derived from an alphavirus, e.g., as described by Pushko et al., [Virology 239(2):389-401 (1997)]. An RP cannot propagate in cell cultures or animal hosts (without a helper plasmid or analogous component), because the replicon does not encode the alphavirus structural components (e.g., capsid and glycoproteins).

The terms “FCV F9-Like” and “F9-Like FCV” are used interchangeably with each other and with the term “classical FCV” and as used herein is an FCV that can be characterized as an older and formerly, universal vaccine strain of FCV, for which the FCV F9 strain is considered a typical representative. In direct contrast, the FCV termed virulent systemic “VS-FCV” or as used herein interchangeably “(VS) FCV”, is a newer class of FCV, which is unusually virulent, and cannot be neutralized by antibodies from the FCV F9-Like strains [see, U.S. Pat. No. 7,449,323; Radford et al., 38(2) Vet res. 319-335 (2007)].

The terms “originate from”, “originates from” and “originating from” are used interchangeably with respect to a given protein antigen and the pathogen or strain of that pathogen that naturally encodes it, and as used herein signify that the unmodified and/or truncated amino acid sequence of that given protein antigen is encoded by that pathogen or strain of that pathogen. The coding sequence, within a nucleic acid construct of the present invention for a protein antigen originating from a pathogen, may have been genetically manipulated so as to result in a modification and/or truncation of the amino acid sequence of the expressed protein antigen relative to the corresponding sequence of that protein antigen in the pathogen or strain of pathogen (including naturally attenuated strains) it originates from.

As used herein, the terms “protecting”, or “providing protection to”, or “eliciting protective immunity to”, “aids in prevention of disease”, and “aids in the protection” do not require complete protection from any indication of infection. For example, “aids in the protection” can mean that the protection is sufficient such that, after challenge, symptoms of the underlying infection are at least reduced, and/or that one or more of the underlying cellular, physiological, or biochemical causes or mechanisms causing the symptoms are reduced and/or eliminated. It is understood that “reduced,” as used in this context, means relative to the state of the infection, including the molecular state of the infection, not just the physiological state of the infection.

As used herein, a “vaccine” is a composition that is suitable for application to an animal, e.g., feline (including, in certain embodiments, humans, while in other embodiments being specifically not for humans) comprising one or more antigens typically combined with a pharmaceutically acceptable carrier such as a liquid containing water, which upon administration to the animal induces an immune response strong enough to minimally aid in the protection from a disease arising from an infection with a wild-type micro-organism, i.e., strong enough for aiding in the prevention of the disease, and/or preventing, ameliorating or curing the disease.

As used herein, a multivalent vaccine is a vaccine that comprises two or more different antigens. In a particular embodiment of this type, the multivalent vaccine stimulates the immune system of the recipient against two or more different pathogens.

The terms “adjuvant” and “immune stimulant” are used interchangeably herein, and are defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance an immune response to one or more vaccine antigens/isolates. Accordingly, “adjuvants” are agents that nonspecifically increase an immune response to a particular antigen, thus reducing the quantity of antigen necessary in any given vaccine, and/or the frequency of injection necessary in order to generate an adequate immune response to the antigen of interest. In this context, an adjuvant is used to enhance an immune response to one or more vaccine antigens/isolates. The American Association of Feline Practitioners Feline Vaccination Guidelines, for example, suggest the use of nonadjuvanted FeLV vaccines [AAFP Feline Advisory Panel, 15: 785-808 (2013)].

As used herein, a “nonadjuvanted vaccine” is a vaccine or a multivalent vaccine that does not contain an adjuvant.

As used herein, the term “pharmaceutically acceptable” is used adjectivally to mean that the modified noun is appropriate for use in a pharmaceutical product. When it is used, for example, to describe an excipient in a pharmaceutical vaccine, it characterizes the excipient as being compatible with the other ingredients of the composition and not disadvantageously deleterious to the intended recipient animal, e.g., feline.

Parenteral administration” includes subcutaneous injections, submucosal injections, intravenous injections, intramuscular injections, intradermal injections, and infusion.

As used herein the term “antigenic fragment” in regard to a particular protein (e.g., a protein antigen) is a fragment of that protein that is antigenic, i.e., capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor. For example, an antigenic fragment of an FCV capsid protein is a fragment of the capsid protein that is antigenic. Preferably, an antigenic fragment of the present invention is immune-dominant for antibody and/or T cell receptor recognition. In particular embodiments, an antigenic fragment with respect to a given protein antigen is a fragment of that protein that retains at least 25% of the antigenicity of the full length protein. In preferred embodiments, an antigenic fragment retains at least 50% of the antigenicity of the full length protein. In more preferred embodiments, an antigenic fragment retains at least 75% of the antigenicity of the full length protein. Antigenic fragments can be as small as 20 amino acids or at the other extreme, be large fragments that are missing as little as a single amino acid from the full-length protein. In particular embodiments, the antigenic fragment comprises 25 to 150 amino acid residues. In other embodiments, the antigenic fragment comprises 50 to 250 amino acid residues. For example, for FeLV, the FeLV gp45 glycoprotein and the FeLV gp70 glycoprotein are antigenic fragments of the FeLV gp85 glycoprotein, whereas, for FCV, one antigenic fragment of an FCV capsid protein comprises region E of the ORF2.

As used herein, one amino acid sequence is 100% “identical” or has 100% “identity” to a second amino acid sequence when the amino acid residues of both sequences are identical. Accordingly, an amino acid sequence is 50% “identical” to a second amino acid sequence when 50% of the amino acid residues of the two amino acid sequences are identical. The sequence comparison is performed over a contiguous block of amino acid residues comprised by a given protein, e.g., a protein, or a portion of the polypeptide being compared. In a particular embodiment, selected deletions or insertions that could otherwise alter the correspondence between the two amino acid sequences are taken into account.

As used herein, nucleotide and amino acid sequence percent identity can be determined using C, MacVector (MacVector, Inc. Cary, N.C. 27519), Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996) and the Clustal W algorithm with the alignment default parameters and default parameters for identity. These commercially available programs can also be used to determine sequence similarity using the same or analogous default parameters. Alternatively, an Advanced Blast search under the default filter conditions can be used, e.g., using the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program using the default parameters.

As used herein, the term “inactivated” microorganism is used interchangeably with the term “killed” microorganism. For the purposes of this invention, an “inactivated” microorganism is an organism which is capable of eliciting an immune response in an animal, but is not capable of infecting the animal. An antigen of the present invention (e.g., an inactivated feline calicivirus) may be inactivated by an agent selected from the group consisting of binary ethyleneimine, formalin, beta-propiolactone, thimerosal, or heat. In a particular embodiment, inactivated feline calicivirus isolates combined with an RP of the present invention are inactivated by binary ethyleneimine.

The alphavirus RNA replicon particles of the present invention may be lyophilized and rehydrated with a sterile water diluent. On the other hand, when the alphavirus RNA replicon particles are stored separately, but intended to be mixed with other vaccine components prior to administration, the alphavirus RNA replicon particles can be stored in the stabilizing solution of those components, e.g., a high sucrose solution.

A vaccine of the present invention can be readily administered by any standard route including intravenous, intramuscular, subcutaneous, oral, intranasal, intradermal, and/or intraperitoneal vaccination. The skilled artisan will appreciate that the vaccine composition is preferably formulated appropriately for each type of recipient animal and route of administration.

Thus, the present invention also provides methods of immunizing a feline against feline pathogens. One such method comprises injecting a feline with an immunologically effective amount of a vaccine of the present invention, so that the feline produces appropriate anti-pathogen antibodies.

Multivalent Vaccines

Accordingly, the present invention provides multivalent vaccines comprising at least one modified live feline pathogen and one or more alphavirus RNA replicon particles. For example, the coding sequence of a protein antigen or antigenic fragment thereof, or combination of such coding sequences of protein antigens useful in a feline vaccine can be added to an alphavirus RNA replicon particle (RP) or combined in the same RP as one that encodes e.g., an FCV capsid protein and/or the FeLV glycoprotein (gp85) in the multivalent vaccine.

In specific embodiments, vaccines comprise at least one modified live feline pathogen and an alphavirus RNA replicon particle that encodes an FCV F9-Like capsid protein or an antigenic fragment thereof, and/or a VS-FCV capsid protein or an antigenic fragment thereof, with another alphavirus RNA replicon particle that encodes an FeLV gp85 or an antigenic fragment thereof. In other embodiments, vaccines comprise at least one modified live feline pathogen and one alphavirus RNA replicon particle that encodes a VS-FCV capsid protein or an antigenic fragment thereof, another alphavirus RNA replicon particle that encodes an FeLV gp85 or an antigenic fragment thereof, and still a third alphavirus RNA replicon particle that encodes an FCV F9-Like capsid protein or an antigenic fragment thereof. In yet other embodiments, vaccines comprise at least one modified live feline pathogen and an alphavirus RNA replicon particle that encodes an FCV F9-Like capsid protein or an antigenic fragment thereof, a VS-FCV capsid protein or an antigenic fragment thereof, and an FeLV gp85 or an antigenic fragment thereof.

Examples of pathogens that one or more of such protein antigens can originate from include feline viral rhinotracheitis Virus (FVR), feline leukemia virus (FeLV), feline panleukopenia Virus (FPL) feline herpesvirus (FHV), other FCV strains, feline parvovirus (FPV), feline infectious peritonitis virus (FIPV), feline immunodeficiency virus, borna disease virus (BDV), rabies virus, feline influenza virus, canine influenza virus, avian influenza, canine pneumovirus, feline pneumovirus, Chlamydophila felis (FKA Chlamydophila psittaci), Bordetella bronchiseptica, and Bartonella spp. (e.g., B. henselae). In particular embodiments, a coding sequence for a capsid protein or analogous protein from one or more of these feline or canine pathogens can be inserted into the same RP as the FCV antigen. Alternatively, or in combination therewith, a coding sequence for a capsid protein or analogous protein from one or more of these feline or canine pathogens can be inserted into one or more other RPs, which can be combined in a vaccine with an RP that encodes the FCV F9-Like capsid protein or an antigenic fragment thereof and/or the VS-FCV capsid protein or an antigenic fragment thereof.

Accordingly, the present invention provides vaccines comprising one or more alphavirus RNA replicon particles (RP) of the present invention [e.g., a VS-FCV capsid protein or an antigenic fragment thereof] along with one or more modified live (attenuated) virus isolates, e.g., a live attenuated older vaccine strain of FCV, such as a live attenuated FCV F9, and/or a live attenuated feline herpesvirus and/or a live attenuated feline parvovirus and/or a live, attenuated feline leukemia virus, and/or a live, attenuated feline infectious peritonitis virus and/or a live, attenuated feline immunodeficiency virus and/or a live, attenuated borna disease virus and/or a live, attenuated rabies virus, and/or a live, attenuated feline influenza virus and/or a live, attenuated canine influenza virus, and/or a live, attenuated avian influenza, and/or a live, attenuated canine pneumovirus, and/or a live, attenuated feline pneumovirus. In addition, a live, attenuated Chlamydophila felis, and/or a live, attenuated Bordetella bronchiseptica and/or a live, attenuated Bartonella spp. (e.g., B. henselae) also can be included in such multivalent vaccines.

Furthermore, the vaccines of the present invention comprising one or more alphavirus RNA replicon particles of the present invention [e.g., encoding a VS-FCV capsid protein or an antigenic fragment thereof] with one or more modified live, virus isolates, further can comprise one or more killed virus isolates such as a killed FCV strain, and/or a killed feline herpesvirus and/or a killed feline parvovirus and/or a killed feline leukemia virus, and/or a killed feline infectious peritonitis virus and/or a killed feline immunodeficiency virus and/or a killed borna disease virus and/or a killed rabies virus, and/or a killed feline influenza virus and/or a killed canine influenza virus, and/or a killed avian influenza virus, and/or a killed canine pneumovirus, and/or a killed feline pneumovirus. In addition, bacterins of Chlamydophila felis, and/or Bordetella bronchiseptica and/or Bartonella spp. (e.g., B. henselae) can also be included in such multivalent vaccines.

It is also to be understood that this invention is not limited to the particular configurations, process steps, and materials disclosed herein as such configurations, process steps, and materials may vary somewhat. It is also to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

SEQUENCE TABLE SEQ ID NO: Description Type  1 Feline Calicivirus nucleic (VS-FCV) acid DNA  2 Feline Calicivirus amino (VS-FCV) acid  3 Feline Calicivirus nucleic (F9-like) acid DNA  4 Feline Calicivirus amino (F9-like) acid  5 FeLV viral glycoprotein nucleic  (gp85) acid DNA  6 FeLV viral glycoprotein amino (gp85) acid  7 FeLV viral glycoprotein nucleic  (gp70) acid DNA  8 FeLV viral glycoprotein amino (gp70) acid  9 Rabies virus Glycoprotein nucleic  acid DNA 10 Rabies virus Glycoprotein amino acid 11 GGCGCGCCGCACC nucleic acid 12 Feline Calicivirus (VS-FCV) nucleic acid RNA 13 Feline Calicivirus (F9-like) nucleic  acid RNA 14 FeLV viral glycoprotein nucleic (gp85) acid RNA 15 FeLV viral glycoprotein nucleic (gp70) acid RNA 16 Rabies virus Glycoprotein nucleic  acid RNA TTAATTAA nucleic acid

SEQUENCES SEQ ID NO: 1 Feline Calicivirus capsid (VS-FCV) atggctgacgacggatctgtgaccaccccagaacaaggaacaatggtcggaggagtgatt gccgaacccagcgctcagatgtcaactgcggcggacatggcctccggaaagtcggtggac tccgagtgggaagccttcttctcgttccacacgtccgtgaactggagcacctccgaaacc caaggaaagatcctcttcaagcagtccctgggtcccctgctgaacccgtacctggagcac atcagcaagctgtacgtcgcttggagcgggtcgatcgaagtgcgattttccatctcggga agcggcgtgttcggtggtaaactggccgccatcgtcgtgccgcctggtgtcgaccctgtc cagtcaacctccatgctgcagtacccgcacgtcctgttcgacgcaagacaagtggagcca gtgatcttctccatcccggacctccgcaacagcctgtatcacttgatgtccgataccgat accacttccctcgtgatcatggtgtacaacgatctgatcaacccgtacgccaatgactcc aacagctcgggttgcatcgtgaccgtcgaaacgaagcctggcatcgatttcaagtttcat ctgctgaaaccgcccggatccatgcttactcacgggtccatcccttccgatctgatcccc aagagctcctccctgtggattgggaaccgccactggaccgatattaccgatttcgtgatt cggcctttcgtgttccaagccaaccggcacttcgacttcaaccaggagactgccggctgg tcaactccacggttccgcccattggccgtgactgtgtcgcagtcaaagggagccaagctc gggaacggcatcgccaccgactacattgtgcctggaatccccgacggatggcctgatact accatccccaccaagctgacccctaccggagattacgccatcacctcctccgacggcaat gatattgaaaccaagctggaatacgagaacgcggacgtgattaagaacaacaccaacttc cgctccatgtatatctgcggaagcctccagagggcttggggcgacaagaagatcagcaac accgggttcatcactaccggagtgatttctgacaactccatcagcccttcgaacacaatt gaccagtccaagatcgtggtgtaccaggacaaccatgtcaattcggaggtccagactagc gacatcactcttgccatcctgggctacaccggaattggagaagaggccataggcgccaac cgggactccgtcgtgagaatttccgtgcttccggaaactggagcaaggggcggaaatcac cccatcttctacaaaaattccatgaagctgggctacgtgatctcctccattgacgtgttc aactcccaaatcctccacacctcgcgccagctgtcactgaacaactacttgttgccccct gactccttcgcggtgtaccggattattgacagcaacggatcatggttcgacattgggatt gacagcgatgggttttcattcgtgggcgtgtcgtcatttccaaagctggagtttccgctg tccgcctcatacatgggcatccagctcgcaaagatccggctggcgtccaacatccggtca tccatgactaagctgtga SEQ ID NO: 2 Feline Calicivirus capsid (VS-FCV) MADDGSVTTPEQGTMVGGVIAEPSAQMSTAADMASGKSVDSEWEAFFSFHTSVNWSTSET QGKILFKQSLGPLLNPYLEHISKLYVAWSGSIEVRFSISGSGVFGGKLAAIVVPPGVDPV QSTSMLQYPHVLFDARQVEPVIFSIPDLRNSLYHLMSDTDTTSLVIMVYNDLINPYANDS NSSGCIVTVETKPGIDFKFHLLKPPGSMLTHGSIPSDLIPKSSSLWIGNRHWTDITDFVI RPFVFQANRHFDFNQETAGWSTPRFRPLAVTVSQSKGAKLGNGIATDYIVPGIPDGWPDT TIPTKLTPTGDYAITSSDGNDIETKLEYENADVIKNNTNFRSMYICGSLQRAWGDKKISN TGFITTGVISDNSISPSNTIDQSKIVVYQDNHVNSEVQTSDITLAILGYTGIGEEAIGAN RDSVVRISVLPETGARGGNHPIFYKNSMKLGYVISSIDVFNSQILHTSRQLSLNNYLLPP DSFAVYRIIDSNGSWFDIGIDSDGFSFVGVSSFPKLEFPLSASYMGIQLAKIRLASNIRS SMTKL (SEQ ID NO: 12) Feline Calicivirus (VS-FCV) capsid auggcugacgacggaucugugaccaccccagaacaaggaacaauggucggaggagugauu gccgaacccagcgcucagaugucaacugcggcggacauggccuccggaaagucgguggac uccgagugggaagccuucuucucguuccacacguccgugaacuggagcaccuccgaaacc caaggaaagauccucuucaagcagucccuggguccccugcugaacccguaccuggagcac aucagcaagcuguacgucgcuuggagcgggucgaucgaagugcgauuuuccaucucggga agcggcguguucggugguaaacuggccgccaucgucgugccgccuggugucgacccuguc cagucaaccuccaugcugcaguacccgcacguccuguucgacgcaagacaaguggagcca gugaucuucuccaucccggaccuccgcaacagccuguaucacuugauguccgauaccgau accacuucccucgugaucaugguguacaacgaucugaucaacccguacgccaaugacucc aacagcucggguugcaucgugaccgucgaaacgaagccuggcaucgauuucaaguuucau cugcugaaaccgcccggauccaugcuuacucacggguccaucccuuccgaucugaucccc aagagcuccucccuguggauugggaaccgccacuggaccgauauuaccgauuucgugauu cggccuuucguguuccaagccaaccggcacuucgacuucaaccaggagacugccggcugg ucaacuccacgguuccgcccauuggccgugacugugucgcagucaaagggagccaagcuc gggaacggcaucgccaccgacuacauugugccuggaauccccgacggauggccugauacu accauccccaccaagcugaccccuaccggagauuacgccaucaccuccuccgacggcaau gauauugaaaccaagcuggaauacgagaacgcggacgugauuaagaacaacaccaacuuc cgcuccauguauaucugcggaagccuccagagggcuuggggcgacaagaagaucagcaac accggguucaucacuaccggagugauuucugacaacuccaucagcccuucgaacacaauu gaccaguccaagaucgugguguaccaggacaaccaugucaauucggagguccagacuagc gacaucacucuugccauccugggcuacaccggaauuggagaagaggccauaggcgccaac cgggacuccgucgugagaauuuccgugcuuccggaaacuggagcaaggggcggaaaucac cccaucuucuacaaaaauuccaugaagcugggcuacgugaucuccuccauugacguguuc aacucccaaauccuccacaccucgcgccagcugucacugaacaacuacuuguugcccccu gacuccuucgcgguguaccggauuauugacagcaacggaucaugguucgacauugggauu gacagcgauggguuuucauucgugggcgugucgucauuuccaaagcuggaguuuccgcug uccgccucauacaugggcauccagcucgcaaagauccggcuggcguccaacauccgguca uccaugacuaagcuguga (SEQ ID NO: 3) Feline Calicivirus (F9-like) capsid atgactgccccggaacaaggaacgatggtcggaggagtgattgcagaaccgtcagcacag atgtccaccgctgccgacatggccactggaaagagcgtggactccgaatgggaagccttc ttctccttccacacttcggtcaactggtcgactagcgaaacccaggggaagattttgttc aagcaatccctcggccctctgctgaacccctacctggagcatctggccaagctgtacgtg gcatggtcgggcagcatcgaagtgcgctttagcatttccggctccggagtgttcggggga aagcttgctgccattgtcgtgccgccaggagtggacccggtgcagtccacttctatgctc caatacccgcatgtcctgttcgacgccagacaggtggagcctgtgatcttttgcctgccg gatctcaggtccaccctgtatcacctcatgtccgacaccgacaccacctcgctcgtgatc atggtgtacaacgacctgatcaacccctacgctaacgacgccaacagctcaggttgcatt gtgactgtcgaaaccaagccaggccctgacttcaagtttcatttgctgaagccgcccggt tccatgctgacccacggctcgatcccatccgacctgatccccaagacgagctccctgtgg atcggaaaccgctactggtccgatattaccgacttcgtgatcagaccattcgtgttccaa gccaaccgccatttcgacttcaaccaggaaaccgcaggatggtcgacccctcgattccgc ccgatttcagtgaccatcaccgaacagaacggcgcgaagctgggaattggcgtggcgacc gactacatcgtgccgggaatcccggatggatggcctgatacgaccattcccggggagctg atccctgccggggactacgccatcaccaacggtactggaaacgacatcaccactgccacc ggttacgacaccgccgacatcataaagaacaacaccaacttcagaggaatgtacatttgc ggctccctgcaacgcgcttggggtgacaaaaagatctcgaacactgccttcatcacaaca gcgactctggacggcgataacaacaacaagatcaatccttgtaataccatcgaccagtcc aaaatcgtggtgttccaggataaccacgtgggaaagaaggcgcagacctccgacgacact ctggcgctgcttggctacaccgggatcggcgagcaggccattggaagcgatcgggatcgg gtcgtgcggatctccaccctccccgagactggagcaaggggaggcaaccaccccatcttt tacaaaaacagcattaagctcggatacgtcatccgctccatcgatgtgttcaactctcaa atcctgcacacttcgcggcagctgtccctgaaccactacctcttgccgcccgactccttc gccgtctaccggatcattgattcgaacgggagctggttcgacatcggcattgatagcgat ggcttctcgtttgtgggcgtgtcgggcttcgggaagctggagttcccactgagcgcctca tacatgggtatccagctggccaagatcaggctggcctccaacatccgctcacctatgact aagctgtga (SEQ ID NO: 4) Feline Calicivirus (F9-like) capsid MTAPEQGTMVGGVIAEPSAQMSTAADMATGKSVDSEWEAFFSFHTSVNWSTSETQGKILF KQSLGPLLNPYLEHLAKLYVAWSGSIEVRFSISGSGVFGGKLAAIVVPPGVDPVQSTSML QYPHVLFDARQVEPVIFCLPDLRSTLYHLMSDTDTTSLVIMVYNDLINPYANDANSSGCI VTVETKPGPDFKFHLLKPPGSMLTHGSIPSDLIPKTSSLWIGNRYWSDITDFVIRPFVFQ ANRHFDFNQETAGWSTPRFRPISVTITEQNGAKLGIGVATDYIVPGIPDGWPDTTIPGEL IPAGDYAITNGTGNDITTATGYDTADIIKNNTNFRGMYICGSLQRAWGDKKISNTAFITT ATLDGDNNNKINPCNTIDQSKIVVFQDNHVGKKAQTSDDTLALLGYTGIGEQAIGSDRDR VVRISTLPETGARGGNHPIFYKNSIKLGYVIRSIDVFNSQILHTSRQLSLNHYLLPPDSF AVYRIIDSNGSWFDIGIDSDGFSFVGVSGFGKLEFPLSASYMGIQLAKIRLASNIRSPMT KL (SEQ ID NO: 13) Feline Calicivirus (F9-like) capsid augacugccccggaacaaggaacgauggucggaggagugauugcagaaccgucagcacag auguccaccgcugccgacauggccacuggaaagagcguggacuccgaaugggaagccuuc uucuccuuccacacuucggucaacuggucgacuagcgaaacccaggggaagauuuuguuc aagcaaucccucggcccucugcugaaccccuaccuggagcaucuggccaagcuguacgug gcauggucgggcagcaucgaagugcgcuuuagcauuuccggcuccggaguguucggggga aagcuugcugccauugucgugccgccaggaguggacccggugcaguccacuucuaugcuc caauacccgcauguccuguucgacgccagacagguggagccugugaucuuuugccugccg gaucucagguccacccuguaucaccucauguccgacaccgacaccaccucgcucgugauc augguguacaacgaccugaucaaccccuacgcuaacgacgccaacagcucagguugcauu gugacugucgaaaccaagccaggcccugacuucaaguuucauuugcugaagccgcccggu uccaugcugacccacggcucgaucccauccgaccugauccccaagacgagcucccugugg aucggaaaccgcuacugguccgauauuaccgacuucgugaucagaccauucguguuccaa gccaaccgccauuucgacuucaaccaggaaaccgcaggauggucgaccccucgauuccgc ccgauuucagugaccaucaccgaacagaacggcgcgaagcugggaauuggcguggcgacc gacuacaucgugccgggaaucccggauggauggccugauacgaccauucccggggagcug aucccugccggggacuacgccaucaccaacgguacuggaaacgacaucaccacugccacc gguuacgacaccgccgacaucauaaagaacaacaccaacuucagaggaauguacauuugc ggcucccugcaacgcgcuuggggugacaaaaagaucucgaacacugccuucaucacaaca gcgacucuggacggcgauaacaacaacaagaucaauccuuguaauaccaucgaccagucc aaaaucgugguguuccaggauaaccacgugggaaagaaggcgcagaccuccgacgacacu cuggcgcugcuuggcuacaccgggaucggcgagcaggccauuggaagcgaucgggaucgg gucgugcggaucuccacccuccccgagacuggagcaaggggaggcaaccaccccaucuuu uacaaaaacagcauuaagcucggauacgucauccgcuccaucgauguguucaacucucaa auccugcacacuucgcggcagcugucccugaaccacuaccucuugccgcccgacuccuuc gccgucuaccggaucauugauucgaacgggagcugguucgacaucggcauugauagcgau ggcuucucguuugugggcgugucgggcuucgggaagcuggaguucccacugagcgccuca uacauggguauccagcuggccaagaucaggcuggccuccaacauccgcucaccuaugacu aagcuguga SEQ ID NO: 5 Feline Leukemia Virus envelope glycoprotein (gp85) atggagtcaccaacacaccctaaaccttctaaagacaaaaccctctcgtggaatctcgccttccttgt gggcatcctgttcacaatcgacatcggcatggccaacccttcgccgcatcagatctacaatgtgacat gggtcattactaatgtgcagacaaacacccaggcaaatgctacttctatgcttggtactctgactgat gcttatccaaccctgcacgtcgacctttgcgatctcgtcggtgacacatgggagcccatcgtgctgaa tccaactaatgtcaaacatggtgccaggtattcttctagcaaatacgggtgtaagaccactgatcgga agaaacagcaacaaacctacccattctacgtgtgcccgggtcacgcaccgtccctgggtccgaaggga acacattgtgggggagcccaagacggtttttgcgctgcttggggttgtgaaacaaccggagaagcctg gtggaagcctacctcatcttgggactacattactgtgaaaagaggctctagccaggataacagctgcg aaggaaagtgtaatcccctggtgcttcaattcacccagaaaggccggcaggcatcatgggatggaccg aaaatgtggggacttagactctatcgcaccggatacgaccccatcgctctgtttactgtgtcacgcca agtctccaccattactccgccacaggccatggggccgaatctggtcctccccgatcagaagccaccct cacggcaaagtcaaaccggctcaaaagtggccacccaacggccccagacaaatgagtccgcacctagg tcagtggcacctacaacaatgggtccaaagcggatcggaaccggagacaggctcattaacctcgtgca agggacttatctggcccttaacgctactgaccccaacaagaccaaggattgctggctctgccttgtga gcagacctccttactatgaggggatcgccattctcggaaactactcaaatcagaccaacccccctccg tcgtgtctgagcaccccccagcacaagcttactatttcagaagtcagtggacagggaatgtgcatcgg aaccgtgccaaagactcatcaagccctttgcaacaaaactcaacaagggcacactggagctcattatc tcgccgcacctaacgggacctactgggcttgcaatactggattgaccccgtgtatctctatggccgtg ctgaattggacttccgacttctgcgtgcttattgagctttggcctagagtgacataccatcagcctga gtacgtctatacccatttcgccaaggcagtcagattccggcgggagcctatctccctgactgtggcct tgatgctcggtggactgacagtgggaggaattgcagctggagtcggaactggaaccaaggccctgctc gaaactgctcagttccggcagctgcagatggccatgcacactgacatccaggctctggaggaatcaat ttcagcccttgagaaaagcttgacctcgctgtctgaagtggtcctccaaaacaggcgcggtttggaca tcctgttccttcaagagggtggtctgtgcgccgctctcaaggaggaatgctgtttctacgctgaccat accgggctggtgcgcgataacatggcaaagctgcgggaacgcttgaaacagaggcagcaactgttcga ctctcagcagggatggttcgagggctggtttaacaagagcccatggtttaccactctgatctcttcaa tcatgggtccactgctcatcctgcttctgattcttctcttcggaccgtgtattctcaacaggctggtg cagtttgtcaaggacagaatctcggtggtccaggccctgattcttactcagcagtatcagcagattaa gcagtacgaccccgatcggccttga SEQ ID NO: 6 Feline Leukemia Virus envelope glycoprotein (gp85) MESPTHPKPSKDKTLSWNLAFLVGILFTIDIGMANPSPHQIYNVTWVITNVQTNTQANAT SMLGTLTDAYPTLHVDLCDLVGDTWEPIVLNPTNVKHGARYSSSKYGCKTTDRKKQQQTY PFYVCPGHAPSLGPKGTHCGGAQDGFCAAWGCETTGEAWWKPTSSWDYITVKRGSSQDNS CEGKCNPLVLQFTQKGRQASWDGPKMWGLRLYRTGYDPIALFTVSRQVSTITPPQAMGPN LVLPDQKPPSRQSQTGSKVATQRPQTNESAPRSVAPTTMGPKRIGTGDRLINLVQGTYLA LNATDPNKTKDCWLCLVSRPPYYEGIAILGNYSNQTNPPPSCLSTPQHKLTISEVSGQGM CIGTVPKTHQALCNKTQQGHTGAHYLAAPNGTYWACNTGLTPCISMAVLNWTSDFCVLIE LWPRVTYHQPEYVYTHFAKAVRFRREPISLTVALMLGGLTVGGIAAGVGTGTKALLETAQ FRQLQMAMHTDIQALEESISALEKSLTSLSEVVLQNRRGLDILFLQEGGLCAALKEECCF YADHTGLVRDNMAKLRERLKQRQQLFDSQQGWFEGWFNKSPWFTTLISSIMGPLLILLLI LLFGPCILNRLVQFVKDRISVVQALILTQQYQQIKQYDPDRP* SEQ ID NO: 14 Feline Leukemia Virus envelope glycoprotein (gp85) auggagucaccaacacacccuaaaccuucuaaagacaaaacccucucguggaaucucgccuuccuugu gggcauccuguucacaaucgacaucggcauggccaacccuucgccgcaucagaucuacaaugugacau gggucauuacuaaugugcagacaaacacccaggcaaaugcuacuucuaugcuugguacucugacugau gcuuauccaacccugcacgucgaccuuugcgaucucgucggugacacaugggagcccaucgugcugaa uccaacuaaugucaaacauggugccagguauucuucuagcaaauacggguguaagaccacugaucgga agaaacagcaacaaaccuacccauucuacgugugcccgggucacgcaccgucccuggguccgaaggga acacauugugggggagcccaagacgguuuuugcgcugcuugggguugugaaacaaccggagaagccug guggaagccuaccucaucuugggacuacauuacugugaaaagaggcucuagccaggauaacagcugcg aaggaaaguguaauccccuggugcuucaauucacccagaaaggccggcaggcaucaugggauggaccg aaaauguggggacuuagacucuaucgcaccggauacgaccccaucgcucuguuuacugugucacgcca agucuccaccauuacuccgccacaggccauggggccgaaucugguccuccccgaucagaagccacccu cacggcaaagucaaaccggcucaaaaguggccacccaacggccccagacaaaugaguccgcaccuagg ucaguggcaccuacaacaauggguccaaagcggaucggaaccggagacaggcucauuaaccucgugca agggacuuaucuggcccuuaacgcuacugaccccaacaagaccaaggauugcuggcucugccuuguga gcagaccuccuuacuaugaggggaucgccauucucggaaacuacucaaaucagaccaaccccccuccg ucgugucugagcaccccccagcacaagcuuacuauuucagaagucaguggacagggaaugugcaucgg aaccgugccaaagacucaucaagcccuuugcaacaaaacucaacaagggcacacuggagcucauuauc ucgccgcaccuaacgggaccuacugggcuugcaauacuggauugaccccguguaucucuauggccgug cugaauuggacuuccgacuucugcgugcuuauugagcuuuggccuagagugacauaccaucagccuga guacgucuauacccauuucgccaaggcagucagauuccggcgggagccuaucucccugacuguggccu ugaugcucgguggacugacagugggaggaauugcagcuggagucggaacuggaaccaaggcccugcuc gaaacugcucaguuccggcagcugcagauggccaugcacacugacauccaggcucuggaggaaucaau uucagcccuugagaaaagcuugaccucgcugucugaagugguccuccaaaacaggcgcgguuuggaca uccuguuccuucaagaggguggucugugcgccgcucucaaggaggaaugcuguuucuacgcugaccau accgggcuggugcgcgauaacauggcaaagcugcgggaacgcuugaaacagaggcagcaacuguucga cucucagcagggaugguucgagggcugguuuaacaagagcccaugguuuaccacucugaucucuucaa ucauggguccacugcucauccugcuucugauucuucucuucggaccguguauucucaacaggcuggug caguuugucaaggacagaaucucggugguccaggcccugauucuuacucagcaguaucagcagauuaa gcaguacgaccccgaucggccuuga SEQ ID NO: 7 Feline Leukemia Virus envelope glycoprotein (gp70) aatcctagtccacaccaaatatataatgtaacttgggtaataaccaatgtacaaactaacacc caagctaacgccacctctatgttaggaaccttaaccgatgcctaccctaccctacatgttgac ttatgtgacctagtgggagacacctgggaacctatagtcctaaacccaaccaatgtaaaacac ggggcacgttactcctcctcaaaatatggatgtaaaactacagatagaaaaaaacagcaacag acataccccttttacgtctgccccggacatgccccctcgttggggccaaagggaacacattgt ggaggggcacaagatgggttttgtgccgcatggggatgtgagaccaccggagaagcttggtgg aagcccacctcctcatgggactatatcacagtaaaaagagggagtagtcaggacaatagctgt gagggaaaatgcaaccccctggttttgcagttcacccagaagggaagacaagcctcttgggac ggacctaagatgtggggattgcgactataccgtacaggatatgaccctatcgctttattcacg gtgtcccggcaggtatcaaccattacgccgcctcaggcaatgggaccaaacctagtcttacct gatcaaaaacccccatcccgacaatctcaaacagggtccaaagtggcgacccagaggccccaa acgaatgaaagcgccccaaggtctgttgcccccaccaccatgggtcccaaacggattgggacc ggagataggttaataaatttagtacaagggacatacctagccttaaatgccaccgaccccaac aaaactaaagactgttggctctgcctggtttctcgaccaccctattacgaagggattgcaatc ttaggtaactacagcaaccaaacaaacccccccccatcctgcctatctactccgcaacacaaa ctaactatatctgaagtatcagggcaaggaatgtgcatagggactgttcctaaaacccaccag gctttgtgcaataagacacaacagggacatacaggggcgcactatctagccgcccccaacggc acctattgggcctgtaacactggactcaccccatgcatttccatggcggtgctcaattggacc tctgatttttgtgtcttaatcgaattatggcccagagtgacttaccatcaacccgaatatgtg tacacacattttgccaaagctgtcaggttccgaaga SEQ ID NO: 8 Feline Leukemia Virus envelope glycoprotein (gp70) NPSPHQIYNVTWVITNVQTNTQANATSMLGTLTDAYPTLHVDLCDLVGDTWEPIVLNPTNVKHGARYSSS KYGCKTTDRKKQQQTYPFYVCPGHAPSLGPKGTHCGGAQDGFCAAWGCETTGEAWWKPTSSWDYITVKRG SSQDNSCEGKCNPLVLQFTQKGRQASWDGPKMWGLRLYRTGYDPIALFTVSRQVSTITPPQAMGPNLVLP DQKPPSRQSQTGSKVATQRPQTNESAPRSVAPTTMGPKRIGTGDRLINLVQGTYLALNATDPNKTKDCWL CLVSRPPYYEGIAILGNYSNQTNPPPSCLSTPQHKLTISEVSGQGMCIGTVPKTHQALCNKTQQGHTGAH YLAAPNGTYWACNTGLTPCISMAVLNWTSDFCVLIELWPRVTYHQPEYVYTHFAKAVRFRR SEQ ID NO: 15 Feline Leukemia Virus envelope glycoprotein (gp70) aauccuaguccacaccaaauauauaauguaacuuggguaauaaccaauguacaaacuaacacc caagcuaacgccaccucuauguuaggaaccuuaaccgaugccuacccuacccuacauguugac uuaugugaccuagugggagacaccugggaaccuauaguccuaaacccaaccaauguaaaacac ggggcacguuacuccuccucaaaauauggauguaaaacuacagauagaaaaaaacagcaacag acauaccccuuuuacgucugccccggacaugcccccucguuggggccaaagggaacacauugu ggaggggcacaagauggguuuugugccgcauggggaugugagaccaccggagaagcuuggugg aagcccaccuccucaugggacuauaucacaguaaaaagagggaguagucaggacaauagcugu gagggaaaaugcaacccccugguuuugcaguucacccagaagggaagacaagccucuugggac ggaccuaagauguggggauugcgacuauaccguacaggauaugacccuaucgcuuuauucacg gugucccggcagguaucaaccauuacgccgccucaggcaaugggaccaaaccuagucuuaccu gaucaaaaacccccaucccgacaaucucaaacaggguccaaaguggcgacccagaggccccaa acgaaugaaagcgccccaaggucuguugcccccaccaccaugggucccaaacggauugggacc ggagauagguuaauaaauuuaguacaagggacauaccuagccuuaaaugccaccgaccccaac aaaacuaaagacuguuggcucugccugguuucucgaccacccuauuacgaagggauugcaauc uuagguaacuacagcaaccaaacaaacccccccccauccugccuaucuacuccgcaacacaaa cuaacuauaucugaaguaucagggcaaggaaugugcauagggacuguuccuaaaacccaccag gcuuugugcaauaagacacaacagggacauacaggggcgcacuaucuagccgcccccaacggc accuauugggccuguaacacuggacucaccccaugcauuuccauggcggugcucaauuggacc ucugauuuuugugucuuaaucgaauuauggcccagagugacuuaccaucaacccgaauaugug uacacacauuuugccaaagcugucagguuccgaaga (SEQ ID NO: 9) RABIES VIRUS G atggtgccgcaggctctcctgtttgtcccccttctggtctttccattgtgttttgggaaattccctatctacacaattc cggacaagttgggaccctggagcccaattgacattcatcatctcagctgcccgaacaatttggtcgtggaggacgaagg atgcaccaacctgtcggggttctcctacatggaattgaaagtcggatacatcagtgccattaagatgaacgggttcact tgcacaggcgtcgtgactgaagctgagacatacactaacttcgtgggatatgtcactaccactttcaaaagaaagcatt tccgccctactcctgatgcttgtagggccgcatacaactggaagatggccggtgaccccagatatgaggaatcacttca caatccgtaccctgactaccactggcttcggactgtcaaaaccaccaaggagtcactcgtgatcattagtccaagtgtg gctgatcttgacccatacgaccggtcacttcactcacgggtgttcccgggggggaattgctctggtgtcgcagtgtcgt caacctactgctccacaaaccacgattacaccatttggatgccagaaaatcctcggcttggtatgtcatgtgacatttt caccaattctcgggggaagagggcttccaaagggtctgaaacttgcggctttgtcgatgagcggggcttgtataagtca cttaaaggtgcttgcaaactcaagctttgtggtgtcttgggattgagattgatggatggaacttgggtcgcaatgcaga cttctaacgaaaccaaatggtgccctcccggacagcttgtgaatttgcatgactttcgctctgacgaaattgagcatct tgtcgtcgaggagttggtcaagaagcgggaagagtgtctggatgctttggaatcaatcatgaccaccaagtcagtgtct ttcagacggctctcacatcttaggaaattggtgccaggttttggaaaagcatataccattttcaacaagacccttatgg aagccgatgctcactacaagtctgtcaggacttggaatgagatcatcccgtctaaagggtgtcttagggtcggagggag atgtcatcctcatgtcaacggagtctttttcaatggtatcattcttggacctgacggaaatgtccttatccctgagatg caatcttccctcctccagcaacacatggaacttcttgtctcatcggtcatcccccttatgcaccccctggctgacccat caaccgtgttcaagaacggtgacgaggcagaggattttgtcgaggtccaccttcccgatgtgcatgaacggatctctgg tgtcgaccttggactccctaactggggaaagtatgtccttctgtcggcaggagccctgactgccttgatgttgattatc ttcctgatgacttgttggaggagagtcaatcggtcggagccaacacaacataatctcagaggaacaggaagggaggtgt cagtcacaccccaaagcgggaagatcatttcgtcttgggagtcatacaagagcggaggtgaaaccggactgtga (SEQ ID NO: 10) RABIES VIRUS G MVPQALLFVPLLVFPLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGF SYMELKVGYISAIKMNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWK MAGDPRYEESLHNPYPDYHWLRTVKTTKESLVIISPSVADLDPYDRSLHSRVFPGGNCSG VAVSSTYCSTNHDYTIWMPENPRLGMSCDIFTNSRGKRASKGSETCGFVDERGLYKSLKG ACKLKLCGVLGLRLMDGTWVAMQTSNETKWCPPGQLVNLHDFRSDEIEHLVVEELVKKRE ECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPS KGCLRVGGRCHPHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLVSSVIPLMHPLAD PSTVFKNGDEAEDFVEVHLPDVHERISGVDLGLPNWGKYVLLSAGALTALMLIIFLMTCW RRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWESYKSGGETGL* (SEQ ID NO: 16) RABIES VIRUS G auggugccgcaggcucuccuguuugucccccuucuggucuuuccauuguguuuugggaaauucccuaucuacacaauuc cggacaaguugggacccuggagcccaauugacauucaucaucucagcugcccgaacaauuuggucguggaggacgaagg augcaccaaccugucgggguucuccuacauggaauugaaagucggauacaucagugccauuaagaugaacggguucacu ugcacaggcgucgugacugaagcugagacauacacuaacuucgugggauaugucacuaccacuuucaaaagaaagcauu uccgcccuacuccugaugcuuguagggccgcauacaacuggaagauggccggugaccccagauaugaggaaucacuuca caauccguacccugacuaccacuggcuucggacugucaaaaccaccaaggagucacucgugaucauuaguccaagugug gcugaucuugacccauacgaccggucacuucacucacggguguucccgggggggaauugcucuggugucgcagugucgu caaccuacugcuccacaaaccacgauuacaccauuuggaugccagaaaauccucggcuugguaugucaugugacauuuu caccaauucucgggggaagagggcuuccaaagggucugaaacuugcggcuuugucgaugagcggggcuuguauaaguca cuuaaaggugcuugcaaacucaagcuuuguggugucuugggauugagauugauggauggaacuugggucgcaaugcaga cuucuaacgaaaccaaauggugcccucccggacagcuugugaauuugcaugacuuucgcucugacgaaauugagcaucu ugucgucgaggaguuggucaagaagcgggaagagugucuggaugcuuuggaaucaaucaugaccaccaagucagugucu uucagacggcucucacaucuuaggaaauuggugccagguuuuggaaaagcauauaccauuuucaacaagacccuuaugg aagccgaugcucacuacaagucugucaggacuuggaaugagaucaucccgucuaaagggugucuuagggucggagggag augucauccucaugucaacggagucuuuuucaaugguaucauucuuggaccugacggaaauguccuuaucccugagaug caaucuucccuccuccagcaacacauggaacuucuugucucaucggucaucccccuuaugcacccccuggcugacccau caaccguguucaagaacggugacgaggcagaggauuuugucgagguccaccuucccgaugugcaugaacggaucucugg ugucgaccuuggacucccuaacuggggaaaguauguccuucugucggcaggagcccugacugccuugauguugauuauc uuccugaugacuuguuggaggagagucaaucggucggagccaacacaacauaaucucagaggaacaggaagggaggugu cagucacaccccaaagcgggaagaucauuucgucuugggagucauacaagagcggaggugaaaccggacuguga

The following examples serve to provide further appreciation of the invention but are not meant in any way to restrict the effective scope of the invention.

EXAMPLES Example 1 Incorporation of the Coding Sequences for FCV Capsid Proteins into the Alphavirus RNA Replicon Particles Introduction

RNA viruses have been used as vector-vehicles for introducing vaccine antigens, which have been genetically engineered into their genomes. However, their use to date has been limited primarily to incorporating viral antigens into the RNA virus and then introducing the virus into a recipient host. The result is the induction of protective antibodies against the incorporated viral antigens. Alphavirus RNA replicon particles have been used to encode pathogenic antigens. Such alphavirus replicon platforms have been developed from several different alphaviruses, including Venezuelan equine encephalitis virus (VEE) [Pushko et al., Virology 239:389-401 (1997)], Sindbis (SIN) [Bredenbeek et al., Journal of Virology 67:6439-6446 (1993) the contents of which are hereby incorporated herein in their entireties], and Semliki Forest virus (SFV) [Liljestrom and Garoff, Biotechnology (NY) 9:1356-1361 (1991), the contents of which are hereby incorporated herein in their entireties]. Moreover, alphavirus RNA replicon particles are the basis for several USDA-licensed vaccines for swine and poultry. These include: Porcine Epidemic Diarrhea Vaccine, RNA Particle (Product Code 19U5.P1), Swine Influenza Vaccine, RNA (Product Code 19A5.D0), Avian Influenza Vaccine, RNA (Product Code 1905.D0), and Prescription Product, RNA Particle (Product Code 9PP0.00).

Alphavirus RNA Replicon Particle Construction

RP-FCV:

Amino acid sequences for FCV capsid proteins were used to generate codon-optimized (feline codon usage) nucleotide sequences in silico. Optimized sequences were prepared as synthetic DNA by a commercial vendor (ATUM, Newark, Calif.). Accordingly, synthetic genes were designed based on the amino acid sequences of a VS-FCV capsid protein and an FCV F9-like capsid protein, respectively. The constructs encoded amino acid sequence [SEQ ID NO: 2] for the VS-FCV capsid protein, or [SEQ ID NO: 4] for the FCV F9-like capsid protein, were codon-optimized for feline, with flanking sequence appropriate for cloning into the alphavirus replicon plasmid.

The VEE replicon vectors designed to express FCV capsid proteins were constructed as previously described [see, U.S. Pat. No. 9,441,247 B2; the contents of which are hereby incorporated herein by reference], with the following modifications. The TC-83-derived replicon vector “pVEK” [disclosed and described in U.S. Pat. No. 9,441,247 B2] was digested with restriction enzymes AscI and PacI. A DNA plasmid containing the codon-optimized open reading frame nucleotide sequence of the FCV capsid genes, with 5′ flanking sequence (5′-GGCGCGCCGCACC-3′) [SEQ ID NO: 11] and 3′ flanking sequence (5′-TTAATTAA-3′), were similarly digested with restriction enzymes AscI and PacI. The synthetic gene cassette was then ligated into the digested pVEK vector.

Production of TC-83 RNA replicon particles (RP) was conducted according to methods previously described [U.S. Pat. No. 9,441,247 B2 and U.S. Pat. No. 8,460,913 B2; the contents of which are hereby incorporated herein by reference]. Briefly, pVHV replicon vector DNA and helper DNA plasmids were linearized with NotI restriction enzyme prior to in vitro transcription using MegaScript T7 RNA polymerase and cap analog (Promega, Madison, Wis.). Importantly, the helper RNAs used in the production lack the VEE subgenomic promoter sequence, as previously described [Kamrud et al., J Gen Virol. 91(Pt 7):1723-1727 (2010)]. Purified RNA for the replicon and helper components were combined and mixed with a suspension of Vero cells, electroporated in 4 mm cuvettes, and returned to OptiPro® SFM cell culture media (Thermo Fisher, Waltham Mass.). Following overnight incubation, alphavirus RNA replicon particles were purified from the cells and media by passing the suspension through a ZetaPlus BioCap depth filter (3M, Maplewood, Minn.), washing with phosphate buffered saline containing 5% sucrose (w/v), and finally eluting the retained RP with 400 mM NaCl buffer. Eluted RP were formulated to a final 5% sucrose (w/v), passed through a 0.22 micron membrane filter, and dispensed into aliquots for storage. Titer of functional RP was determined by immunofluorescence assay on infected Vero cell monolayers.

RP-FeLV:

An amino acid sequence for FeLV gp85 were used to generate codon-optimized (feline codon usage) nucleotide sequences in silico. Optimized sequences were prepared as synthetic DNA by a commercial vendor (ATUM, Newark, Calif.). Accordingly, a synthetic gene was designed based on the amino acid sequence of gp85. The construct (gp85_wt) was wild-type amino acid sequence [SEQ ID NO: 2], codon-optimized for feline, with flanking sequence appropriate for cloning into the alphavirus replicon plasmid.

The VEE replicon vectors designed to express FeLV gp85 were constructed as previously described [see, U.S. Pat. No. 9,441,247 B2; the contents of which are hereby incorporated herein by reference in their entireties], with the following modifications. The TC-83-derived replicon vector “pVEK” [disclosed and described in U.S. Pat. No. 9,441,247 B2] was digested with restriction enzymes AscI and PacI. A DNA plasmid containing the codon-optimized open reading frame nucleotide sequence of the FeLV gp85 genes, with 5′ flanking sequence (5′-GGCGCGCCGCACC-3′) [SEQ ID NO: 11] and 3′ flanking sequence (5′-TTAATTAA-3′), was similarly digested with restriction enzymes AscI and PacI. The synthetic gene cassette was then ligated into the digested pVEK vector, and the resulting clone was re-named “pVHV-FeLV gp85”. The “pVHV” vector nomenclature was chosen to refer to pVEK-derived replicon vectors containing transgene cassettes cloned via the AscI and PacI sites in the multiple cloning site of pVEK.

Production of TC-83 RNA replicon particles (RP) was conducted according to methods previously described [U.S. Pat. No. 9,441,247 B2 and U.S. Pat. No. 8,460,913 B2; the contents of which are hereby incorporated herein by reference in their entireties]. Briefly, pVHV replicon vector DNA and helper DNA plasmids were linearized with NotI restriction enzyme prior to in vitro transcription using MegaScript T7 RNA polymerase and cap analog (Promega, Madison, Wis.). Importantly, the helper RNAs used in the production lack the VEE subgenomic promoter sequence, as previously described [Kamrud et al., J Gen Virol. 91(Pt 7):1723-1727 (2010)]. Purified RNA for the replicon and helper components were combined and mixed with a suspension of Vero cells, electroporated in 4 mm cuvettes, and returned to OptiPro® SFM cell culture media (Thermo Fisher, Waltham Mass.). Following overnight incubation, alphavirus RNA replicon particles were purified from the cells and media by passing the suspension through a ZetaPlus BioCap depth filter (3M, Maplewood, Minn.), washing with phosphate buffered saline containing 5% sucrose (w/v), and finally eluting the retained RP with 400 mM NaCl buffer. Eluted RP were formulated to a final 5% sucrose (w/v), passed through a 0.22 micron membrane filter, and dispensed into aliquots for storage. Titer of functional RP was determined by immunofluorescence assay on infected Vero cell monolayers.

RP-RV

A vaccine was prepared comprising an alphavirus RNA replicon particle encoding the rabies virus glycoprotein (G) from rabies virus (RV) packaged with the capsid protein and glycoproteins of the avirulent TC-83 strain of Venezuelan Equine Encephalitis Virus. The nucleotide sequence for the rabies virus G protein was codon-optimized for humans. The resulting sequence has only ˜85% nucleotide identity to a live rabies virus glycoprotein (G) sequence, despite having 100% amino acid identity. The vaccine can be used as a single dose administered to a mammalian subject, e.g., subcutaneously to cats and dogs aged 12 weeks or older or alternatively, in a multiple dose comprising a primary administration followed by one or more booster administrations.

An amino acid sequence for Rabies glycoprotein (G) was used to generate codon-optimized (human codon usage) nucleotide sequences in silico. Optimized sequences were prepared as synthetic DNA by a commercial vendor (ATUM, Newark, Calif.). Accordingly, a synthetic gene [SEQ ID NO: 9] was designed based on the amino acid sequence of Rabies Glycoprotein. The construct (RABV-G) was a wild-type amino acid sequence [SEQ ID NO: 10], codon-optimized for humans, with flanking sequence appropriate for cloning into the alphavirus replicon plasmid.

The VEE replicon vectors that were designed to express Rabies G were constructed as previously described [see, U.S. Pat. No. 9,441,247 B2; the contents of which are hereby incorporated herein by reference], with the following modifications. The TC-83-derived replicon vector “pVEK” [disclosed and described in U.S. Pat. No. 9,441,247 B2] was digested with restriction enzymes AscI and PacI. A DNA plasmid containing the codon-optimized open reading frame nucleotide sequence of the Rabies G gene, with 5′ flanking sequence (5′-GGCGCGCCGCACC-3′) [SEQ ID NO: 11] and 3′ flanking sequence (5′-TTAATTAA-3′) was similarly digested with restriction enzymes AscI and PacI. The synthetic gene cassette was then ligated into the digested pVEK vector, and the resulting clone was re-named “pVHV-RABV-G”. The “pVHV” vector nomenclature was chosen to refer to pVEK-derived replicon vectors containing transgene cassettes cloned via the AscI and PacI sites in the multiple cloning site of pVEK.

Production of TC-83 RNA replicon particles (RP) was conducted according to methods previously described [U.S. Pat. No. 9,441,247 B2 and U.S. Pat. No. 8,460,913 B2; the contents of which are hereby incorporated herein by reference in their entireties]. Briefly, pVHV replicon vector DNA and helper DNA plasmids were linearized with NotI restriction enzyme prior to in vitro transcription using MegaScript T7 RNA polymerase and cap analog (Promega, Madison, Wis.). Importantly, the helper RNAs used in the production lack the VEE subgenomic promoter sequence, as previously described [Kamrud et al., J Gen Virol. 91(Pt 7):1723-1727 (2010)]. Purified RNA for the replicon and helper components were combined and mixed with a suspension of Vero cells, electroporated in 4 mm cuvettes, and returned to OptiPro® SFM cell culture media (Thermo Fisher, Waltham, Mass.). Following overnight incubation, alphavirus RNA replicon particles were purified from the cells and media by passing the suspension through a ZetaPlus BioCap® depth filter (3M, Maplewood, Minn.), washing with phosphate buffered saline containing 5% sucrose (w/v), and finally eluting the retained RP with 400 mM NaCl buffer. Eluted RP were formulated to a final 5% sucrose (w/v), passed through a 0.22 micron membrane filter, and dispensed into aliquots for storage. Titer of functional RP was determined by immunofluorescence assay on infected Vero cell monolayers.

Example 2 Evaluation of the Safety of a Combination Vaccine Containing Two RP Constructs and Three Modified Live Fractions in Cats

Various formulations and reconstitution methods of a lyophilized pentavalent feline combination vaccine were evaluated in regard to their safety in cats. The desired presentation of the pentavalent vaccine is a 0.5 mL dose, the optimal formulation and fill method is a 0.5 mL fill. The stabilizer contained 1.1% NZ-amine (a casein enzymatic hydrolysate), 1.1% gelatin, and 7.5% sucrose, with the percentages provided representing the final concentrations. Should the volume and final potency constraints for the addition of the five fractions necessitate a volume greater than 0.5 mL, the product can be formulated and filled to 1.0 mL and reconstituted with 0.5 mL of diluent. This doubles the concentration of stabilizer components in the administered dose. The safety of such a concentrated dose of stabilizer with these antigens had not previously been tested in cats.

A third option of a 1.0 mL fill volume rehydrated with 1.0 mL of diluent also was tested in the event that the 1.0 mL fill/0.5 mL rehydration format was not safe in cats. The vaccines were blended and then lyophilized. The vaccines contained an alphavirus RNA replicon particle that encodes a feline leukemia virus glycoprotein (RP-FeLV) and an alphavirus RNA replicon particle that encodes a feline calicivirus capsid protein (RP-FCV), together with the components of a commercially available vaccine, Nobivac® Feline-1, i.e., a modified live (MLV) feline panleukopenia virus (FPL), a modified live feline viral rhinotracheitis virus (FVR) and a modified live Chlamydophila felis in a stabilizer containing 1.1% gelatin, 1.1% NZ-amine and 7.5% sucrose. The various formulations of the pentavalent feline combination vaccine were prepared and reconstituted as described in the Table 1 below:

TABLE 1 FORMULATIONS OF THE PENTAVALENT COMBINATION VACCINE Freeze-Dried Diluent Treatment No. of Test Product Cake (Fill) (Rehydration) Group Cats Antigens Volume Volume 1 6 RP-FCV, 1.0 mL 1.0 mL 2 6 RP-FeLV, FPL, 1.0 mL 0.5 mL 3 6 FVR & C. felis 0.5 mL 0.5 mL 4 2 Diluent only NA 1.0 mL

The vaccines were formulated to contain the same dose of each antigen (the 0.5 mL cake vaccine was formulated with twice the concentration of each antigen), the stabilizer was used at a constant concentration in all formulations (the 1.0 mL cake vaccine rehydrated with 0.5 mL diluent contained twice the concentration of stabilizer upon rehydration).

The experimental cat subjects were vaccinated with the indicated volume of the respective test vaccine at 7-8 weeks of age (typically having a minimum age of vaccination for the feline core vaccine) and again 21 days later. The cats were observed for a period of 15 minutes immediately after each vaccination for reactions to the test vaccine, which might indicate pain or discomfort, such as vocalization, stinging, itching, biting, sudden movement upon the injection of the vaccine, or any unusual reaction (see Tables 2 and 3 below). A clinical assessment was performed by a veterinarian 4 to 6 hours after the vaccination and each day for three days post-vaccination.

The clinical assessment included palpation of the injection site and observations for any local reactions including pain to touch, swelling, redness or abscess. Cats were also observed for any systemic reactions such as depression, lethargy, lameness, vomiting, tremors, agitation, and diarrhea. Body temperatures were also measured and recorded at 4 to 6 hours post-vaccination and each day for two days following each vaccination. Palpation of injection sites for local reactions was additionally performed three times each week between 7 days and 21 days after each vaccination.

All vaccine formulations and rehydration protocols were found to be safe. No local or systemic reactions were observed in any of the cats. Body temperatures at each measurement period for all cats were normal, with the exception of one cat in Treatment Group 3 (0.5 mL cake/0.5 mL diluent) which presented with a body temperature of 103.6° C. five hours post-second vaccination. Therefore, all three preparations of the pentavalent vaccine were found to be acceptable.

TABLE 2 IMMEDIATE REACTIONS TO VACCINATION WITH PENTAVALENT COMBINATION VACCINE Sudden Scratching movement at injection Bite (during away from site (post- Vocalization injection) injection injection) Treatment 1^(st) 2^(nd) 1^(st) 2^(nd) 1^(st) 2^(nd) 1^(st) 2^(nd) Group Vaccine ID Vac. Vac. Vac. Vac. Vac. Vac. Vac. Vac. 1 Pentavalent Feline 0/6 1/6 0/6 0/6 0/6 0/6 0/6 0/6 Vaccine 1.0 mL cake/ 1.0 mL diluent 2 Pentavalent Feline 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 Vaccine 1.0 mL cake/ 0.5 mL diluent 3 Pentavalent Feline 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 Vaccine 0.5 mL cake/ 0.5 mL diluent 4 Diluent only 1/2 1/2 0/2 0/2 1/2 1/2 0/2 0/2 1.0 mL

TABLE 3 LOCAL/SYSTEMIC REACTIONS TO VACCINATION WITH PENTAVALENT COMBINATION VACCINE Systemic Reactions Local (vomiting, Reactions lameness, (Redness, depression, pain to lethargy, Body touch, tremors, Temperature swelling agitation or ≥103.5° C. or abscess) diarrhea) Post-Vaccination Treatment 1^(st) 2^(nd) 1^(st) 2^(nd) 1^(st) 2^(nd) Group Vaccine ID Vac. Vac. Vac. Vac. Vac. Vac. 1 Pentavalent 0/6 0/6 0/6 0/6 0/6 0/6 Feline Vaccine 1.0 mL cake/ 1.0 mL diluent 2 Pentavalent 0/6 0/6 0/6 0/6 0/6 0/6 Feline Vaccine 1.0 mL cake/ 0.5 mL diluent 3 Pentavalent 0/6 0/6 0/6 0/6 0/6 1/6 Feline Vaccine 0.5 mL cake/ 0.5 mL diluent 4 Diluent only 0/2 0/2 0/2 0/2 0/2 0/2 1.0 mL

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. 

We claim:
 1. A vaccine to aid in the prevention of disease due to feline calicivirus (FCV) comprising an alphavirus RNA replicon particle that encodes one or more FCV capsid proteins and a modified live feline pathogen selected from the group consisting of a modified live feline viral rhinotracheitis virus (FVR), a modified live feline panleukopenia virus (FPLV), a modified live Chlamydophila felis, and any combination thereof, and a pharmaceutically acceptable carrier.
 2. The vaccine of claim 1, wherein the one or more FCV capsid proteins is selected from the group consisting of a FCV F9-Like capsid protein, a virulent systemic FCV (VS-FCV) capsid protein, or both a FCV F9-Like capsid protein and a virulent systemic FCV (VS-FCV) capsid protein.
 3. The vaccine of claim 2, wherein the alphavirus RNA replicon particle is a Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particle.
 4. A method of immunizing a feline against a pathogenic FCV comprising administering to the feline an immunologically effective amount of the vaccine of claim
 3. 5. The vaccine of claim 3, wherein the VEE alphavirus RNA replicon particle also encodes an antigen selected from the group consisting of a feline leukemia virus (FeLV) glycoprotein gp85, a FeLV glycoprotein gp70, a FeLV glycoprotein gp45, a rabies virus G protein, or any combination thereof.
 6. The vaccine of claim 5, wherein the antigen is selected from the group consisting of an FeLV gp85 that comprises an amino acid sequence comprising at least 95% identity with the amino acid sequence of SEQ ID NO: 6, an FeLV gp70 that comprises an amino acid sequence comprising 95% identity or more with the amino acid sequence of SEQ ID NO: 8, and the combination thereof.
 7. The vaccine of claim 5, wherein the antigen is a rabies virus G protein that comprises an amino acid sequence comprising at least 95% identity with the amino acid sequence of SEQ ID NO:
 10. 8. The vaccine of claim 3, that further comprises an additional VEE alphavirus RNA replicon particle that encodes an antigen selected from the group consisting of a feline leukemia virus (FeLV) glycoprotein gp85, a FeLV glycoprotein gp70, a FeLV glycoprotein gp45, a rabies virus G protein, or any combination thereof.
 9. The vaccine of claim 8, wherein the antigen is selected from the group consisting of an FeLV gp85 that comprises an amino acid sequence comprising at least 95% identity with the amino acid sequence of SEQ ID NO: 6, an FeLV gp70 that comprises an amino acid sequence comprising 95% identity or more with the amino acid sequence of SEQ ID NO: 8, and the combination thereof.
 10. The vaccine of claim 8, wherein the antigen is a rabies virus G protein that comprises an amino acid sequence comprising at least 95% identity with the amino acid sequence of SEQ ID NO:
 10. 11. The vaccine of claim 3, wherein the FCV capsid protein is a VS-FCV capsid protein that comprises an amino acid sequence comprising at least 95% identity with the amino acid sequence of SEQ ID NO:
 2. 12. The vaccine of claim 3, wherein the FCV capsid protein is a FCV F9-Like capsid protein that comprises an amino acid sequence comprising at least 95% identity with the amino acid sequence of SEQ ID NO:
 4. 13. A vaccine to aid in the prevention of disease due to FCV comprising a Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particle that encodes a virulent systemic feline calicivirus (VS-FCV) capsid protein, a modified live feline viral rhinotracheitis virus (FVR), a modified live feline panleukopenia virus (FPLV), a modified live Chlamydophila felis and a pharmaceutically acceptable carrier; wherein the VS-FCV capsid protein comprises an amino acid sequence comprising at least 95% identity with the amino acid sequence of SEQ ID NO:
 2. 14. A method of immunizing a feline against a pathogenic FCV comprising administering to the feline an immunologically effective amount of the vaccine of claim
 13. 15. The vaccine of claim 13, wherein the VEE alphavirus RNA replicon particle further encodes a feline calicivirus F9-Like capsid protein; wherein the FCV F9-Like capsid protein comprises an amino acid sequence comprising at least 95% identity with the amino acid sequence of SEQ ID NO:
 4. 16. The vaccine of claim 15, further comprising a Venezuelan Equine Encephalitis (VEE) alphavirus RNA replicon particle that encodes a feline leukemia virus (FeLV) glycoprotein gp85, a FeLV glycoprotein gp70, or both; wherein the FeLV gp85 comprises an amino acid sequence comprising at least 95% identity with the amino acid sequence of SEQ ID NO: 6 and the FeLV gp70 comprises an amino acid sequence comprising 95% identity or more with the amino acid sequence of SEQ ID NO:
 8. 17. A method of immunizing a feline against a pathogenic FCV comprising administering to the feline an immunologically effective amount of the vaccine of claim
 16. 18. A method of immunizing a feline against a pathogenic FCV comprising administering to the feline an immunologically effective amount of the vaccine of claim
 15. 19. The vaccine of claim 13, further comprising a VEE alphavirus RNA replicon particle that encodes a feline calicivirus F9-Like capsid protein; wherein the FCV F9-Like capsid protein comprises an amino acid sequence comprising at least 95% identity with the amino acid sequence of SEQ ID NO:
 4. 20. A method of immunizing a feline against a pathogenic FCV comprising administering to the feline an immunologically effective amount of the vaccine of claim
 19. 